The technical field of this invention relates to methods of decellularizing an isolated organ or part of an organ, by mechanically agitating the isolated organ with a fluid that removes the cellular membrane surrounding the isolated organ, and with a fluid that solubilizes the cytoplasmic and nuclear components of the isolated organ.
Techniques for restoring structure and function to damaged organs or tissue are used routinely in the area of reconstructive surgery. For example, artificial materials for replacing limbs and teeth. (See e.g. Paul (1999), J. Biomech, 32: 381-393; Fletchall, et al., (1992) J. Burn Care Rehabil, 13: 584-586 and Wilson et al., (1970) Artif. Limbs, 14: 53-56).
Tissue transplantation is another way of restoring function by replacing the damaged organ, and has saved the lives of many. However, problems exist when there is a transfer of biological material form one individual to another. Organ rejection is a significant risk associated with transplantation, even with a good histocompatability match. Immunosuppressive drugs such as cyclosporin and FK506 are usually given to the patient to prevent rejection. These immunosuppressive drugs however, have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression can weaken the immune system, which can lead to a threat of infection. In some instances, even immunosuppression is not enough to prevent organ rejection. Another major problem of transplantation, is the availability of donor organs. In the United States alone there are about 50,000 people on transplant waiting lists, many of whom will die before an organ becomes available.
Due to these constraints, investigators are involved in the technology of producing artificial organs in vitro for in vivo transplantation. The artificial organs typically are made of living cells fabricated onto a matrix or a scaffold made of natural or manmade material. These artificial organs avoid the problems associated with rejection or destruction of the organ, especially if the subject""s own tissue cells are used for reconstruction of the artificial organ. These artificial organs also avoid the problem of not having enough donor organs available because any required number of organs can be reconstructed in vitro.
Vacanti et al have disclosed methods for culturing cells in a three-dimensional polymer-cell scaffold made of a biodegradable polymer. Organ cells are cultured within the polymer-cell scaffold which is implanted into the patient. Implants made of resorbable materials are suggested for use as temporary replacements, rather than a permanent replacement. The object of the temporary replacement is to allow the healing process to replace the resorbed material. Naughton et al. reported a three-dimensional tissue culture system in which stromal cells were laid over a polymer support system (See U.S. Pat. No. 5,863,531).
The above methods however, rely on shaping the support scaffold into the desired configuration of the organ. Shaping the matrix scaffold involves one of many procedures, such as solvent casting, compression, moulding, and leaching. These techniques do not always result in a matrix shape scaffold that is the same size as a native in vivo organ requiring replacement. A correct three-dimensional configuration is essential for the reconstructed organ to function properly in vivo. Not only is the shape required to fit into the body cavity, but the shape also creates the necessary microenvironment for the cultured cells to attach, proliferate, differentiate and in some cases, migrate through the matrix scaffold. These critical requirements can be met by the choice of the appropriate material of the scaffold and also be effected by the processing techniques. Optimal cell growth and development arises when the interstitial structure of the microenvironment resembles the interstitial structure of a natural organ.
The shaping process may have deleterious effects on the mechanical properties of scaffold, and in many cases produce scaffolds with irregular three-dimensional geometries. Additionally, many shaping techniques have limitations that prevent their use for a wide variety of polymer materials. For example, poly L-lactic acid (PLLA) dissolved in methylene chloride and cast over the mesh of polyglycolic acid (PGA) fibers is suitable for PGA, however, the choice of solvents, and the relative melting temperatures of other polymers restricts the use of this technique for other polymers. Another example includes solvent casting, which is used for a polymer that is soluble in a solvent such as chloroform. The technique uses several salt particles that are dispersed in a PLLA/chloroform solution and cast into a glass container. The salt particles utilized are insoluble in chloroform. The solvent is allowed to evaporate and residual amounts of the solvent are removed by vacuum-drying. The disadvantages of this technique is that it can only be used to produce thin wafers or membranes up to 2 mm in thickness. A three-dimensional scaffold cannot be constructed using this technique.
Due to the limitations of the shaping techniques, and due to the importance of having a scaffold with the correct three-dimensional shape, a need exists for producing a decellularized organ that has the same three-dimensional interstitial structure, shape and size as the native organ. Reconstruction of an artificial organ using a decellularized organ will produce an artificial organ that functions as well as a native organ, because it retains the same shape, size and interstitial structure which enables the deposited cells to resume a morphology and structure comparable to the native organ.
In general, the invention pertains to methods of producing decellularized organs, using an isolated organ or a part of an organ and a series of extractions that removes the cell membrane surrounding the organ, or part of an organ, and the cytoplasmic and nuclear components of the isolated organ, or part of an organ.
Accordingly, in one aspect, the invention provides a method for producing a decellularized organ comprising:
mechanically agitating an isolated organ to disrupt cell membranes without destroying the interstitial structure of the organ;
treating the isolated organ in a solubilizing fluid at a concentration effective to extract cellular material from the organ without dissolving the interstitial structure of the organ; and
washing the isolated organ in a washing fluid to remove cellular debris without removing the interstitial structure of the organ until the isolated organ is substantially free of cellular material, to thereby produce a decellularized organ.
The method can further comprise equilibrating the decellularized organ in an equilibrating fluid. The equilibrating fluid can be selected from the group consisting of distilled water, physiological buffer and culture medium. The method can further comprise drying the decellularized organ. The dried decellularized organ can be stored at a suitable temperature, or equilibrated in a physiological buffer prior to use.
In one embodiment, the step of mechanically agitating the isolated organ further comprises placing the isolated organ in a stirring vessel having a paddle which rotates at a speed ranging from about 50 revolutions per minute (rpm) to about 150 rpm.
In one embodiment, the step of mechanically agitating the isolated organ occurs in a fluid selected from the group consisting of distilled water, physiological buffer and culture medium.
In one embodiment, the step of treating the isolated organ in the solubilizing fluid also occurs in a stirring vessel. In a preferred embodiment, the solubilizing fluid is an alkaline solution having a detergent. In more preferred embodiment, the alkaline solution is selected from the group consisting of sulphates, acetates, carbonates, bicarbonates and hydroxides, and a detergent is selected from the group consisting of Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), polyoxyethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W-1 (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl xcex2P-D-glucopuranoside, n-heptyl xcex2-D glucopyranoside, n-Octyl xcex1-D-glucopyranoside and Nonidet P-40. In the most preferred embodiment, the solubilizing solution is an ammonium hydroxide solution having Triton X-100.
In one embodiment, the step of washing the isolated organ also occurs in a stirring vessel. The washing fluid can be selected from the group consisting of distilled water, physiological buffer and culture medium.
In another aspect, the invention features a method for producing a decellularized kidney comprising:
mechanically agitating an isolated kidney in distilled water to disrupt cell membranes without destroying the interstitial structure of the kidney;
treating the isolated kidney in an alkaline solution having a detergent at a concentration effective to extract cellular material without dissolving the interstitial structure of the kidney;
washing the isolated kidney in distilled water to remove cellular debris without removing the interstitial structure of the kidney until the kidney is substantially free of the cellular material, to thereby produce a decellularized kidney.
In a preferred embodiment, the method further comprises equilibrating the decellularized kidney in a phosphate buffered solution. In another embodiment, the method further comprises drying the decellularized kidney. Embodiments for mechanically agitating a decellularized organ are described above and are reiterated here. In another preferred embodiment, the step of washing further comprises rotating the isolated kidney in distilled water in a stirring vessel.